1. Field of the Invention
The present invention relates to the fields of human diseases and disorders, more specifically to methods of selectively homing and delivering an agent to human cells, combining targeting specificity with intracellular triggered release of the payload. The present invention can be applied, for example, in the treatment and/or diagnosis of different types of cancer as well as other diseases and disorders.
2. Background Information
Amongst human diseases and disorders, cancer is the leading cause of death throughout the western world. Even though, in most cases, diagnosis is often followed by surgery and the prognosis is favourable, recurrent forms often appear after surgery, indicating that metastases were already present by the time the disease was detected. This is the major obstacle for a complete remission of the tumor.
Traditional chemotherapy constitutes one of the most important treatment modalities against cancer. Chemotherapeutic agents, upon systemic administration, are generally characterized by a high volume of distribution that leads to a poor selectivity towards tumor cells and accumulation in healthy tissues. Such pattern of distribution can lead to increased toxicities against normal tissues that also show enhanced proliferative rates, such as the bone marrow, gastrointestinal tract and hair follicles. Myelosuppression, alopecia or mucositis are examples of some of the most unpleasant and undesired consequences of fighting cancer with conventional therapy (Ferrara, 2005). Side effects that occur as a result of toxicities to normal tissues mean that anticancer chemotherapeutics are often given at sub-optimal doses, resulting in the eventual failure of therapy. This is often accompanied by the development of drug resistance and metastatic disease. Targeted drug delivery towards tumor cells, on the other hand, offers the possibility of overcoming these consequences by directing and concentrating the therapeutic agent only at the desired target site, increasing therapeutic efficacy through increased tumor cell death and decreased incidence of side effects in healthy tissues (Allen, 2002).
Tumor cells require a dedicated and effective blood supply, which cannot be provided by the existing vessels in normal tissues (Folkman, 1990). Therefore, angiogenesis, a process common on wound healing, starts to develop in order to create an appropriate blood vessel network to irrigate the novel cellular mass. Since angiogenesis is controlled by pro- and anti-angiogenic factors, it appears to be a promising target in cancer therapy (Ferrara, 2005). Angiogenic vessels present distinct features at different levels, mainly on the markers expressed at the cell surface (Carmeliet, 2003). Many of these tumor vessel markers are proteins associated with tumor-induced angiogenesis and some are specific for certain tumors (Pasqualini, 2002).
Targeting therapeutic agents to the vasculature of tumors, as opposed to the tumor cells themselves, offers some additional advantages: eliminating tumor's blood supply can profoundly suppress tumor growth; blood vessels are more readily accessible to intravenously administered therapy than tumor cells, and although tumor blood vessels acquire a tumor-associated ‘signature’, they are composed of normal cells that do not readily acquire mutations that could further lead to drug resistance (Boehm, 1997); in addition, tumor vascular targeting avoids problems associated with intrinsic drug resistance such as those related with poor drug penetration into a tumor due to high interstitial pressure gradients within tumors (Feron, 2004). Treatment selectivity against proliferative tumor-derived endothelial cells and minimal toxicity is likely to be achieved because angiogenesis in the adult is limited to wound healing, ovulation, pregnancy and atherosclerosis (Folkman, 2007; Folkman, 2005; Hanahan, 1996).
In general terms, treatment selectivity can be achieved by designing a system where the agent is concealed, whereas the surface is decorated in a way that it has the ability to direct the system to the target site, taking advantage of one or more distinct features of the pathological site. In this regard, one of the most important strategies in molecularly guided cancer pharmacology is the development of techniques that can modify the kinetic features of drugs by encapsulating them in nanosystems, like liposomes.
The development of physically and biological stable liposomes, composed with a hydrophilic polymer, like poly(ethylene glycol), PEG, on its surface, with an average size of 100 nm and containing chemotherapeutic drugs, such as doxorubicin, was a significant achievement that, presumably, will have a great impact in the future of nanotechnology, within the field of human health. Coating the surface of liposomes with a hydrophilic polymer like PEG, strongly contributes to the formation of a hydrophilic cloud around the liposomes (Needham, 1992 #44; Woodle, 1992 #39; Hristova, 1995 #45; Hajitou, 2006 #62). Upon intravenous injection, such hydrophilic shell dramatically decreases the rate and the extent of electrostatic and hydrophobic interactions between the surface of liposomes and blood components that mediate liposomal blood clearance and/or disintegration (Lasic, 1991; Needham, 1992; Torchilin, 1994; Woodle, 1992). The ability of drug-loaded PEG-grafted liposomes to long circulate in blood, favours their accumulation in solid tumors (Wu, 1993). Such accumulation, as well as that of macromolecules or polymeric drugs is greatly enhanced in tumor tissue relative to that in healthy tissues, a phenomenon known as Enhanced Permeability and Retention, being generally observed in viable and rapidly growing solid tumors (Maeda, 2001). This phenomenon is supported by an extensive angiogenesis and impaired lymphatic drainage at the tumor interstitium (Maeda, 2000). Tumor vessels possess irregular cellular lining composed of disorganized, loosely connected, branched or overlapping endothelial cells, which contribute to tumor vessel leakiness (Carmeliet, 2003; Hashizume, 2000). As an example, it was previously demonstrated that PEG-grafted liposomes, following transendothelial transport through gaps between endothelial cells, presented significant extravascular accumulation in tumors (Yuan, 1994). Further improvements in the selective toxicity of anti-proliferative drugs might be achieved by coupling ligands selective for the target cell to the liposome surface. Relatively few ligand molecules per liposome (10-20) are required to selectively deliver high payloads of drugs to target cells via the mechanism of receptor-mediated internalization (Allen, 2002). Unlike other delivery systems such as drug-immunoconjugates or -immunotoxins, which deliver few molecules of drug or toxin (<10) per antibody (or immunotoxin) molecule, ligand-targeted liposomes can be exploited to deliver thousands of molecules of drug using few tens of molecules of ligands covalently coupled on the liposome surface (Sapra, 2003). Coupling a ligand to a support should be a simple, fast, efficient and reproducible method, yielding stable, non-toxic bonds. Moreover, the coupling reaction should not alter the drug loading efficiency, drug release rates, nor the biological properties of the ligands, e.g. target recognition and binding efficiency (Papahadjopoulos, 1991).
The versatility of liposomes as a delivery system allows the control of the location (spatial delivery) as well as of the rate of release (temporal delivery) of the transported agent. pH-sensitive liposomes constitute a typical example where both spatial and temporal delivery can be achieved. They are usually composed of a neutral cone-shaped lipid like dioleoylphosphatidylethanolamine (DOPE) and a weakly acidic amphiphile, such as cholesteryl hemisuccinate (CHEMS), and designed to form a stable lipid bilayer at neutral or basic pH but to rapid destabilize in an acidifying endosome (Fonseca, 2005). Since pH-sensitive liposomes can facilitate cytosolic release of membrane impermeable molecules, it might be feasible to combine their use with a targeting ligand that promotes receptor-mediated endocytosis. Overall, the development of a sterically stabilized pH-sensitive nanosystem covalently coupled to a targeting ligand, able to target specific cells, like tumor cells and/or endothelial cells existing in tumor blood vessels, containing a chemotherapeutic drug, such as doxorubicin, can have a major impact on the therapeutic index of the encapsulated payload, in the treatment of diseases like human breast cancer.